Sex and estradiol influence glial pro-inflammatory responses to lipopolysaccharide in rats
Lisa C. LoramPaige W. SholarFrederick R. TaylorJulie L. WieslerJessica A. BabbKeith A. StrandDebra BerkelhammerHeidi E.W. DaySteven F. MaierLinda R. Watkins
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Neuroglia
Sexual dimorphism
Toll-like receptor 4 (TLR4), especially expressed on monocytes/macrophages, connects microbial and sterile innate immune activation. Lipopolysaccharide (LPS) from Gram-negative bacteria and several endogenous molecules, among others saturated fatty acids (SFAs), are able to induce signalling through this receptor. Downstream inflammatory cytokines orchestrate the immune response. Our aim was to investigate how long-lasting multifactorial stress affects Gram-negative signalling and search for possible correlations between cytokine production and TLR4 expression or SFA concentration.Eight healthy males were studied during a 7-day ranger-training course with semi-continuous physical strain, together with energy and sleep restrictions. Blood drawn on days 0, 3, 5 and 7 was incubated ex vivo for 6 h with or without LPS 10 ng/mL, whereupon surface expression of TLR4 on CD14⁺ monocytes and supernatant concentrations of inflammatory cytokines (TNF-α, IL-1β and IL-6) were measured. In addition, plasma free fatty acids were quantified.Monocyte TLR4 expression was elevated throughout the course (p < 0.05 vs. baseline). Corresponding results were found for SFAs. The concentration of TNF-α increased significantly on day 3 and thereafter normalized, and a similar pattern was seen for IL-1β. No correlations were found between cytokine concentrations and monocyte TLR4 expression or plasma SFAs.Multifactorial stress significantly affected ex vivo production of TNF-α and monocyte surface expression of TLR4. In addition, mobilization of fat resulted in increased plasma concentrations of SFAs. No associations between inflammatory cytokines and monocyte TLR4 expression or SFAs were found.
Monocyte
Ex vivo
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Abstract LPCAT2 is a lipid-modifying enzyme that co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response; however, its effect on TLR4 co-receptor–CD14 is unknown. RAW264.7 cells, a common murine macrophage experimental model, were used to study the effect of LPCAT2 on CD14 expression. The expression of LPCAT2 in RAW264.7 cells was silenced using RNA interference and treated with 100ng/ml of various lipopolysaccharide chemotypes. We found that CD14 expression induced by smooth lipopolysaccharide was significantly decreased (p < 0.05) in RAW264.7 macrophages with LPCAT2 silenced. This study suggests that LPCAT2 regulates CD14 gene and protein expression. This implies that LPCAT2 can regulate CD14-dependent cellular activities.
Lipid raft
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Neonatal Sepsis
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The lipopolysaccharide (LPS) receptor complex consists of two interacting receptors (CD14 and TLR4) and an associated protein (MD-2). When engaged by LPS, as in gram-negative infection, this complex transduces a signal detected by MyD88 and passed onward by a cascade of the IRAKs, TRAF6, and NIK, resulting in activation of NF-κB. A similar cascade, mediated by TLR2, occurs with ligands derived from gram-positive bacteria. In vitro studies of human monocytes have shown that TLR4 mRNA is paradoxically upregulated in response to "tolerizing" doses of LPS. This study evaluated changes in vivo of blood monocyte CD14, TLR4, TLR2, and MD-2 mRNA by reverse transcription followed by real-time polymerase chain reaction in surgical intensive care unit patients and in normal controls. In addition cell-surface receptor expression of TLR2, TLR4, and CD14 was assessed by flow cytometry in patients and normal controls. Inflammation-induced acute tolerance to LPS was evaluated by ex vivo whole blood tumor necrosis factor α production and was significantly reduced in patients compared with controls, confirming LPS hyporesponsiveness. Monocyte mRNA and cell-surface receptor expression of TLR4 were increased 2.4-fold (P < 0.05) and 1.7-fold (P < .002), respectively, in patients compared with normal controls. Monocyte TLR2 mRNA, MD-2 mRNA and CD14 and TLR2 cell-surface expression were not significantly changed compared with controls. The present study suggests that the acute inflammatory condition associated with peripheral cellular LPS hyporesponsiveness is neither specific to prior infectious challenge nor can be ascribed to significant alterations in expression of the cell-surface LPS binding complex proteins.
Monocyte
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Abstract : The term sepsis describes a potentially lethal clinical condition that develops as a result of a dysregulated host response to bacterial infection. The most common bacterial component implicated in initiating the septic syndrome is a cell wall molecule derived from Gram‐negative bacteria, known as lipopolysaccharide (LPS) or endotoxin. Like all mammals, humans are equipped with an LPS‐sensing machinery consisting, primarily, of LPS‐binding protein (LBP), CD14, a glycosylphosphatidylinositol (GPI)‐anchored monocyte differentiation antigen, and toll‐like receptor 4 (TLR4), a signal‐transducing integral membrane protein. Modest stimulation of TLR4 facilitates the elimination of invading microorganisms. Potent TLR4 stimulation, however, produces severe reactions in the host, often leading to multiple organ failure and death. The search for pharmaceuticals that reduce mortality in septic patients has been a painstaking process. Thus far, only a few compounds have been found to significantly reduce mortality rates. Perhaps one of the more promising therapeutic strategies currently pursued is based on the identification of synthetic or naturally occurring substances that neutralize LPS or inhibit LPS‐mediated activation of host immune cells, such as monocytes and macrophages. Here, we describe a number of diverse molecular structures with a capacity to either enhance or blunt LPS‐induced monocyte activation. The underlying molecular mechanisms are discussed.
Monocyte
Lipid A
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Objective To explore the effect of LPS on the expression of CD14 and Toll like receptor 4 (TLR4) in rat Kupffer cells (KCs).Methods In the rat KCs stimulated with LPS,the expression of CD14,TLR4,TNFα and IL 6 was detected.Results LPS could obviously up regulated the expression of CD14 and TLR4 in a dose dependent manner (LPS 10?mg/L,CD14/GAPDH=0.73,TLR4/GAPDH=0.63) and time dependent manner (0.5 24.0?h,the expression of CD14 reached the peak during 3 to 6 h,CD14/GAPDH=0.86).The expression of CD14 and TLR4 in KCs stimulated by the active mediators from KCs which had been exposed to LPS 1 h was obviously increased.Conclusion There were a close correlation between LPS or the active mediators from KCs induced by LPS and the expression of CD14,TLR 4.It is implied that the increases of TLR4 and CD14 expression may be induced by LPS in 1 3?h,and later further increases of TLR4 and CD14 expression may be correlated with the cytokines produced by KCs.
Glyceraldehyde 3-phosphate dehydrogenase
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Bacterial endotoxin [lipopolysaccharide (LPS)] is essential for bacterial virulence as it has a biphasic effect which is either harmful and leads to aseptic shock and death or assists the body defense mechanisms as it stimulates B-cells activation. Many studies have noted that LPS do their action through activation of CD14/ TLR4 pathways, which occur mainly in liver cells, including Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells, which are responsible for cytokines releases and shows the good or bad LPS effect.
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Monocyte
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Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) and other exogenous and endogenous molecules, and is thought to contribute to defense mechanisms against infections. Our objective was to elucidate the clinical significance of TLR4 in acute infectious diseases by analyzing its sequential expression on CD14+ monocytes. Peripheral blood samples were obtained from 36 patients with acute infectious diseases on admission and after treatment within certain intervals. The TLR4 expression on CD14+ monocytes was analyzed using flow cytometry and was presented as a mean fluorescence intensity (MFI). TLR4 expression during the acute phase of infection was highly enhanced compared to that of normal subjects (MFI: 22.1 vs 8.5). TLR4 expression was promptly reduced to normal levels in parallel with the disease improvement. In patients who died despite treatment, the enhancement of TLR4 expression during the acute phase was less prominent compared to those who survived (MFI: 14.6 vs 23.5) and its sequential change was also subtle. These results indicate that monocytes respond to acute infections by the induction of TLR4 expression and that a poor response may be associated with a poor prognosis.
Monocyte
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Abstract: Bradykinin‐ and substance P (SP)‐stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin‐stimulated synthesis of phospho‐ inositides (PI) in type‐1 astrocytes and oligodendrocytes. SP‐stimulated PI accumulation was restricted to oligoden‐ drocyte/type‐2 astrocyte progenitor cells and type‐2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP‐stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type‐2 astrocyte type‐2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.
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